The primary objective of this proposal is to determine the roles of polysialic acid, NCAM, and HNK-1 glycan in neural development and cancer. In the past few years, we have made critical progress in this field. First, we showed that the polysialyltransferases STSSia II and STSSia IV synergistically synthesize highly dense and longer polysialic acid on NCAM than either one of the polysialyltransferases alone. Second, we showed that STSSia ll-deficient mice generated by homologous recombination exhibit misguided infrapyramidal mossy fibers in the hippocampus, resulting in higher exploratory drive and reduced behavioral response to fear conditioning without accompanying an anomaly in long term potentiation at hippocampus CAS area. Third, we have generated mutant mice with deficient STSSia IV through homologous recombination and crossbred with STSSia IV deficient mice. We discovered that mice doubly deficient in STSSia II and STSSia IV mostly die within one month after birth and exhibit significantly impaired neural development, exhibiting a thin cerebral cortex, immature cerebellum, small olfactory bulb, and enlarged lateral ventricles. Fourth, we showed that polysialic acid-positive C6 glioma cells are more invasive in the host mouse brain than polysialic acid-negative C6 glioma cells. Fifth, we have generated HNK-1 sulfotransferase deficient mice and found that mutant male mice exhibit infertility in a C57BL/6 genetic background. Based on these critical findings, two major studies are proposed as follows: 1) Determining the roles of polysialic acid and NCAM in neural development. We will determine the specific neural impairment exhibited by mutant mice with doubly deficient STSSia II and STSSia IV or doubly deficient NCAM and STSSia II or STSSia IV. We will also determine whether polysialylation of NCAM is the major contributor to NCAM function by expressing NCAM mutant transgenes in NCAM deficient mice. 2) Determining the roles of HNK-1 glycan in development and cancer. We will determine howthe loss of HNK-1 glycan leads to impairment of testis development. We will also determine if HNK-1 glycan in the brain plays a role in glioma invasion. These studies should lead to development of potential therapeutic strategies for treatment of patients where neural cell migration is overly increased such as occurs in gliomas or neural cell migration is impaired, i.e., after injury.